Primer extension detection methods on active electronic microarrays

• US 20050136441A1
• Filed: 09/15/2004
• Published: 06/23/2005
• Est. Priority Date: 04/12/1999
• Status: Abandoned Application

• Current Assignee: John J. Carrino, Jonathan M. Diver, Louis O. Gerrue
• Original Assignee: John J. Carrino, Jonathan M. Diver, Louis O. Gerrue
• Inventors: Diver, Jonathan M., Carrino, John J., Gerrue, Louis O.
• Application Number:
  US10/942,565
• Publication Number:
  US 20050136441A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: B01J 2219/00317 Microwell devices, i.e. having large numbers of wells
• CPC Class Codes: B01J 2219/00585 Parallel processes
• CPC Class Codes: B01J 2219/00596 Solid-phase processes
• CPC Class Codes: B01J 2219/00605 the compounds being directly bound or immobilised to solid supports
• CPC Class Codes: B01J 2219/00614 Delimitation of the attachment areas
• CPC Class Codes: B01J 2219/00626 Covalent
• CPC Class Codes: B01J 2219/0063 Other, e.g. van der Waals forces, hydrogen bonding
• CPC Class Codes: B01J 2219/00637 by coating it with another layer
• CPC Class Codes: B01J 2219/00644 the porous medium being present in discrete locations, e.g. gel pads
• CPC Class Codes: B01J 2219/00653 the compounds being bound to electrodes embedded in or on the solid supports
• CPC Class Codes: B01J 2219/00659 Two-dimensional arrays
• CPC Class Codes: B01J 2219/00702 Processes involving means for analysing and characterising the products
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: C12Q 1/6825 Nucleic acid detection involving sensors
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 2531/113 PCR
• CPC Class Codes: C12Q 2535/125 Allele specific primer extension
• CPC Class Codes: C12Q 2565/501 being an array of oligonucleotides
• CPC Class Codes: C40B 40/06 Libraries containing nucleotides or polynucleotides, or derivatives thereof
• CPC Class Codes: C40B 60/14 : for creating libraries
• CPC Class Codes: Y10T 436/143333 : Saccharide [e.g., DNA, etc.]

Abstract

This invention relates to methods for detecting the extent of hybridization of a nucleic acid in a sample to a probe nucleic acid sequence by electronically hybridizing the nucleic acid in the sample to the probe, utilizing the hybridized nucleic acid as a template in a nucleic acid polymerase reaction to extend the bound probe and incorporate a labeled nucleotide, and detecting the labeled product. This invention also relates to methods of detecting the extent of hybridization of a plurality of nucleic acids in a sample to a plurality of nucleic acid probes using similar methods.

Claims

1. A method of detecting the extent of hybridization of a nucleic acid in a sample to a probe nucleic acid sequence, the method comprising:

(a) electronically hybridizing the nucleic acid in a sample to a nucleic acid probe bound to a support at a predetermined location;

(b) utilizing the hybridized nucleic acid as a template in a nucleic acid polymerase reaction to extend the bound probe, whereby a labeled nucleotide is incorporated into the extended probe; and

(c) detecting the labeled nucleotide incorporated into the extended bound probe at the predetermined location.

2. The method of claim 1, wherein the labeled nucleotide comprises a labeling moiety selected from the group consisting of fluorescent moieties, colorigenic moieties, chemiluminescent moieties, and affinity moieties.

3. The method of claim 2, wherein the labeled nucleotide comprises a fluorescent moiety.

4. The method of claim 1, wherein the nucleic acid polymerase reaction is a DNA polymerase reaction.

5. The method of claim 1, wherein the nucleic acid polymerase reaction is a reverse-transcriptase reaction.

6. The method of claim 1, wherein a control sequence probe is also bound to the support at the location.

7. The method of claim 3, wherein the fluorescent moiety is selected from the group consisting of cyanine dye moieties, Bodipy Texas Red moieties, rhodamine moieties, fluorescein moieties, and cumarin moieties.

8. A method of detecting the extent of hybridization of a plurality of nucleic acids in a sample to a plurality of nucleic acid probes, the method comprising:

(a) electronically hybridizing the nucleic acid in the sample to a plurality of nucleic acid probes bound to a support at a two or more predetermined locations, wherein;

i) the nucleic acids in the sample are electronically hybridized to nucleic acid probes at two or more locations on the support, and

ii) the sequence of at least one nucleic acid probe on a first location is different from the sequence of the nucleic acid probes on a second location.

(b) utilizing the hybridized nucleic acids as a templates in a nucleic acid polymerase reaction to extend the bound probes, whereby a labeled nucleotide is incorporated into the extended probes; and

(c) detecting the labeled nucleotide incorporated into the extended bound probes at the predetermined location.

9. The method of claim 8, wherein the labeled nucleotide comprises a labeling moiety selected from the group consisting of fluorescent moieties, colorigenic moieties, chemiluminescent moieties, and affinity moieties.

10. The method of claim 9, wherein the labeled nucleotide comprises a fluorescent moiety.

11. The method of claim 8, wherein the fluorescent moiety is selected from the group consisting of cyanine dye moieties, Bodipy Texas Red moieties, rhodamine moieties, fluorescein moieties, and cumarin moieties.

12. The method of claim 11, wherein the fluorescent moiety is a cyanine dye moiety selected from the group consisting of Cy5 and Cy3.

13. The method of claim 8, wherein the nucleic acid polymerase reaction is a DNA polymerase reaction.

14. The method of claim 8, wherein the nucleic acid polymerase reaction is a reverse-transcriptase reaction.

15. The method of claim 8, wherein the two or more locations each comprise at least one nucleic acid probe with the same sequence.

16. The method of claim 15, wherein the nucleic acid probe with the same sequence is a control sequence probe.

17. The method of claim 8, wherein the nucleic acids in the sample are electronically hybridized to nucleic acid probes at five or more locations on the support, and the sequence of at least one nucleic acid probe at each of the five locations is different from the sequences of the nucleic acid probes at the other locations.

18. The method of claim 8, wherein the nucleic acids in the sample are electronically hybridized to nucleic acid probes at ten or more locations on the support, and the sequence of at least one nucleic acid probe at each of the ten locations is different from the sequences of the nucleic acid probes at the other locations.

19. The method of claim 8, wherein the nucleic acids in the sample are electronically hybridized to nucleic acid probes at twenty or more locations on the support, and the sequence of at least one nucleic acid probe at each of the twenty locations is different from the sequences of the nucleic acid probes at the other locations.

20. The method of claim 8, wherein the nucleic acids in the sample are electronically hybridized to nucleic acid probes at forty or more locations on the support, and the sequence of at least one nucleic acid probe at each of the forty locations is different from the sequences of the nucleic acid probes at the other locations.

21. The method of claim 10, wherein the fluorescent moiety is a cyanine dye moiety selected from the group consisting of Cy5 and Cy3.